The present invention relates to gp105, a N-glycosylated glycoprotein substantially purified from other proteins that has a glycosylated molecular weight of 105 kDa and a non-glycosylated molecular weight of 85 kDa, and that is present on the surface of BL3 hematopoietic stem cells but absent from those of other cells including 32D and FDC-P1 myeloid progenitor cells, EL4 T-cells and 3T3 fibroblasts. The invention further relates to methods of using antibody against gp105 to produce an enriched hematopoietic stem cell population.
All circulating blood cells develop from pluripotent stem cells through the process of hematopoiesis. Hematopoietic stem cells are undifferentiated cells capable of self-renewal and differentiation into committed progenitor cells of the myeloid, erythroid, megakaryocytic and lymphoid blood cell lineages. A thorough analysis of hematopoietic stem cells is fundamental to a comprehensive understanding of the developmental biology of the hematolymphoid system. Relatively little is known, however, about hematopoietic stem cells.
Functionally, hematopoietic stem cells are capable of long-term reconstitution of the hematolymphoid system of lethally-irradiated recipients in vivo. See Spangrude & Johnson, PNAS 87:7433-7437 (1990); Spangrude et al., Blood 78:1395-1402 (1991). They also can differentiate into pre-day 12 spleen colony-forming units (CFU-S), which can be observed in in vivo assays for spleen foci formation. See Spangrude et al., Science 241:58-62 (1988); Molineux et al., Exp. Hematol. 14:710 (1986); Nakahata & Ogawa, PNAS 79:3843-3847 (1982). In addition, hematopoietic stem cells develop a "cobblestone" morphology upon adherence in vitro to a layer of stromal cells. See Wong et al., Immunity 1:571-583 (1994).
Efforts to characterize hematopoietic stem cells in more detail have been hampered primarily because of the proportionately minute amount (10.sup.-4 to 10.sup.-5) of hematopoietic stem cells as compared with all cells, even in blood cell-forming organs such as bone marrow or the fetal liver. See Orlic et al., Blood 82:762-770 (1993). Accordingly, the elucidation of physical characteristics unique to hematopoietic stem cells is desirable as a means to produce enriched stem cell populations. See, e.g., Spangrude et al., Blood 78:1395-1402 (1991). All known hematopoietic stem cell enrichment protocols involve cell-separation methods based on the selection for cell surface markers or genetic (retroviral) markers. See Jordan et al., Science 252:953-963 (1990). Although methods of producing enriched populations of hematopoietic stem cells have been described, the absence of unique markers has precluded the isolation of an unequivocally pure population of hematopoietic stem cells.
Hematopoietic stem cells express cell surface differentiation antigen (Thy-1) and stem cell antigen-1 (Sca-1). They do not, however, express the lineage markers (Lin) characteristic of B cells (B220), granulocytes (Gr-1), myelomonocytic cells (Mac-1) and T cells (CD4, CD8)). See Spangrude et al., supra. The reportedly most widely used hematopoietic stem cell enrichment protocol involves the use of monoclonal antibodies against Thy-1 and Sca-1. See Orlic et al., supra. Only a subset, however, of Thy-1.sup.+, Sca-1.sup.+ and Lin.sup.- cells are able to repopulate lethally-irradiated recipients long-term. See Smith et al., PNAS 88:2788-2792 (1991). Selection based on Thy-1 and Sca-1 expression thus does not produce a pure hematopoietic stem cells population. Similarly, other hematopoietic stem cell enrichment techniques such as those which involve the use of monoclonal antibodies against protein tyrosine kinases such as the W locus gene product, c-kit, and fetal liver kinase-2 (flk-2) apparently are unable to distinguish between hematopoietic stem cells and progenitor cells. See, e.g., Matthews et al., Cell 65:1143-1152 (1991).
The recent establishment of a cell line from a lethally-irradiated recipient mouse reconstituted with fetal liver cells previously transduced with a rearranged retroviral genome has been reported. See Wong et al., supra. BL3 cells exhibit all of the functional hematopoietic stem cell properties, i.e., they can reconstitute lethally-irradiated recipients long-term, they give rise to pre-CFU-S and colony-forming cells and they develop "cobblestones" upon association with stromal cells. In addition to being Thy-1.sup.+, Sca-1.sup.+ and Lin.sup.-, BL3 cells also express a transcription factor, GATA-1, known to be expressed in hematopoietic stem cells. See Sposi et al., PNAS 89:6353-6357 (1992). Furthermore, BL3 cells are embryonic in origin, having derived from fetal liver cells of 12-day old mouse embryos. BL3 cells thus may possess different cell surface markers than adult hematopoietic stem cells. See Jordan et al., supra; Spangrude et al., supra.
The foregoing discussion reveals the need to identify other cell surface markers on hematopoietic stem cells, specifically to enable the production of more highly enriched hematopoietic stem cell populations, and generally to facilitate a better understanding of the growth and differentiation of immature blood cells.